The overall objective of this proposal is to shed light on some of the basic questions concerning the biology of the new class I proteins. Because these genes were discovered after cloning and sequencing all of the class I homologous sequences isolated from a human cell line, no antibodies or sera specifically reactive with the protein products of these genes are known. While expression can be detected at the mRNA level in various cells and tissues, protein products have only been detected in HLA class I deficient cell lines transfected with these genes. One characteristic of the classical HLA-A,-B and -C antigens of importance to transplantation biology is the striking polymorphism found at each locus. The first specific aim of this proposal is to determine if, like the classical antigens, the HLA-E, F(5.4) and G(6.0) antigens exhibit polymorphism. We will use the polymerase chain reaction to specifically amplify E, F and G cDNA and genomic sequences from a set of genetically diverse individuals and determine their nucleotide sequence. If polymorphism is present at one or more of these loci, we will use the same methods to correlate that polymorphism with the outcome of an unrelated bone marrow transplant. The second specific aim is to isolate monoclonal antibodies specifically reactive with the HLA-E, -F(5.4) and G(6.0) molecules. We have constructed a set of cell lines expressing these proteins and are planning construction of additional lines. We will use these cells as a source of antigen for the production of monoclonal antibodies. In order to optimize the possibility of producing specific antibodies we are using transgenic mice expressing human beta2 microglobulin and HLA-B27 for immunization. The third specific aim is to determine if the HLA-E and G(6.0) gene products are recognized by T cells. We will first attempt to generate T cell clones reactive with transfectants of the class I deficient LCL .221 expressing the HLA-E and G(6.0) proteins. If polymorphism exists, we will attempt to demonstrate alloreactivity using a cell line from one individual expressing an allo E or G(6.0) antigen using an in vitro CML test to generate T cell clones reactive with the alloantigen. Learning the phenotype of these clones as well as their reactivity with a panel of transfected cell lines will be a fundamental step towards understanding the function of HLA-E and G(6.0). In specific aim four we will investigate the potential of these antigens to function as histocompatibility determinants. This work will help to answer whether these antigens contribute to the problems encountered in transplantation. This study will also provide important information and reagents which will significantly add to our knowledge of the expression and function of these new class I molecules.